The present invention relates to the use of biologically active compounds selected from GH, analogues thereof and GH-releasing compounds, as lipid lowering agents for the preparation of a drug for the treatment of mammals with homozygous familial hypercholesterolemia. By the furnishing of a drug comprising compounds selected from GH, analogues thereof and GH-releasing compounds, optionally in combination with further lipid lowering treatment, elevated plasma cholesterol in LDLR deficient mammals with familial hypercholesterolemia of the homozygous form may be treated.
Growth hormone (GH) has pleiotropic effects on cholesterol metabolism. GH stimulates the expression of hepatic low density lipoprotein (LDL)-receptors and the activity of cholesterol 7xcex1-hydroxylase (C7xcex1OH), a key regulatory step in bile acid synthesis. According to the present invention it is shown that GH treatment reduces plasma cholesterol in the situation of homozygous familial hypercholesterolemia as represented by the recently developed LDL-receptor knockout mouse strain.
GH infusion into LDL-receptor knockout mice resulted in a 30-40% reduced plasma cholesterol level. In addition, the reduced enzymatic activities of cholesterol 7xcex1-hydroxylase and HMG CoA reductase were normalized. It is concluded that GH treatment reduces the severe homozygous form of familial hypercholesterolemia in LDLR-deficient mice. Such therapy gives a beneficial effect in the heavily therapy resistant disease homozygous familial hypercholesterolemia, a disorder known to be strongly resistant to lipid-lowering treatment.
In this specification and the appended claims the following abbreviations are used: C7xcex1OH, represents cholesterol 7xcex1 hydroxylase; HMG CoA reductase, represents 3-hydroxy-3-methyl-glutaryl coenzyme A reductase; FPLC, represents fast performance liquid chromatography; GH, represents growth hormone; HDL, represents high density lipoprotein; LDL, represents low density lipoprotein; LDLR, represents low density lipoprotein receptor; LDLRKO, represents low density lipoprotein receptor knockout; SDS-PAGE, represents sodium dodecyl sulphate-polyacrylamide gel electrophoresis; TNA, represents total nucleic acid; VLDL, represents very low density lipoprotein.
Familial hypercholesterolemia (FH) is a common autosomal dominant inherited disease and is present in heterozygous and homozygous forms.
Heterozygous FH occurs at a frequency of approx. 1:300-500 in the general population. The subjects have type II-A lipid pattern and approximately twice the normal low-density lipoprotein (LDL) cholesterol levels. Heterozygotes have an increased risk to develop premature heart disease and their expected life span is reduced 10 to 15 years. FH heterozygotes have a mutation in the gene encoding the LDL receptor. This receptor is located on the surface of cells in the liver and other organs. The LDL receptors bind LDL and facilitate its uptake by receptor-mediated endocytosis and subsequent delivery to lysosomes, where the LDL is degraded yielding free cholesterol for cellular use. When LDL receptors are deficient, the rate of removal of LDL from plasma declines, and the level of LDL rises in inverse proportions to the receptor number. The excess plasma LDL is deposited in scavenger cells and other cell types, producing atheromas and xanthomas. FH heterozygotes have one normal and one mutant allele at the LDL receptor locus; hence their cells are able to bind and degrade LDL at approximately half the normal rate. (See In: The metabolic and molecular bases of inherited disease. Seventh edition, Mac Graw-Hill, Chapter 62, Familial Hypercholesterolemia, by J. L. Goldstein et al., pp 1981-2030).
Subjects with the homozygous form of FH (incidence=1:106) have plasma cholesterol levels 3-5 fold higher than normal subjects and frequently develop coronary heart disease in childhood, almost invariably before 20 years of age. Homozygotes possess two mutant alleles at the LDL receptor locus, and their cells show a total or near total inability to bind or take up LDL.
There are two animal models for FH that closely resemble the human disease. The first is a natural mutant strain of rabbits, Watanabe Heritable Hyperlipidemic (WHHL) rabbits, and the second is the recently available mouse LDLR knock-out (LDLRKO) strain developed by Herz et al (Ishibashi, S. et al. 1993. J. Clin. Invest. 92; 883-893). Previous studies in WHHL-rabbits have shown that homozygous animals have strongly suppressed activity of the regulatory enzyme C7xcex1OH (Xu et al. 1995. J. Clin. Invest. 95; 1447-1504).
In the therapy of type II-A hyperlipidemia, the strategy is based on the concept of increasing the number of available hepatic LDL-receptors which in turn will reduce plasma cholesterol due to an increased hepatic uptake of LDL and LDL precursor lipoproteins, such as intermediate density lipoprotein, (IDL). In heterozygous FH, the most effective therapy is by interfering with the enterohepatic circulation of bile acids by orally administered bile acid sequestrants such as cholestyramine in combination with specific 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG CoA) reductase inhibitors. An increasing number of such drugs (statins and vastatins) have been introduced to the market. Such combined therapy can reduce, but does seldom completely normalize plasma LDL.
However, in homozygotes that do not harbor any functional LDL receptors, other means to reduce the high LDL levels must be employed. This disease type is a candidate for gene therapy. Such treatment has been tested in laboratory animals using the LDLR gene and the gene encoding cholesterol 7xcex1-hydroxylase (C7xcex1OH), the key regulatory step in the synthesis of bile acids. (Ishibashi, S. et al. 1993. J. Clin. Invest. 92, 883-893, Spady, D. K. and Cuthbert, J. A. 1995. J. Clin. Invest 96; 700-709). The effects are unfortunately transient. So far, the only available therapy has been liver transplantation or plasmapheresis and/or selective extracorporeal removal of cholesterol-rich LDL-particles. The latter therapy, if performed every 2-3 weeks, can reduce but not normalize the increased plasma cholesterol level. Recently it has been shown that certain FH homozygotes can also benefit from high dose statin therapy (Marais, A. D et al. 1997. J. Lipid Res. 38:2071-8). Therefore, until gene-therapy will be clinically available, there is a need of novel pharmacological therapeutic strategies in homozygous FH.
Pituitary growth hormone (GH) has several effects on cholesterol and lipid metabolism. We have previously shown that GH therapy has a stimulatory effect on the hepatic LDLR expression in both rats and humans (Rudling, M. et al. Proc. Natl. Acad. Sci. USA. 1992, 89; 6983-6987), and in particular we have identified GH as an important stimulator of the enzymatic activity of C7xcex1OH (Rudling et al, 1997, J. Clin. Invest. 99; 2239-2245). We have shown that GH stimulates C7xcex1OH activity, not only in hypophysectomized animals, but also in normal young rats (P. Parini, et al. 1999, Cholesterol and lipoprotein metabolism in aging: reversal of hypercholesterolemia by growth hormone treatment in old rats, Arterioscl. Thromb. and Vasc. Biol. 19;832-839). The experiments were performed in mammals expressing normal LDL receptors.
From xe2x80x9cCurrent opinion in Lipidologyxe2x80x9d, 1997,Vol. 8, p. 337-341 by B. Angelin as well as from xe2x80x9cMetabolismxe2x80x9d, 1996, Vol. 45 No. 11, p. 1414-1421 by Tostad et al. it was evident that familial hypercholesterolemia of the heterozygous form may benefit from treatment with GH because such patients express functional LDL receptors.
However, because homozygotes do not harbor functional LDL receptors, it was not for a man skilled in the art obvious that the homozygous form of the disease may benefit from GH treatment.
We have now surprisingly found that administration of compounds selected from GH, analogues thereof and GH-releasing compounds, optionally in combination with established lipid-lowering treatment, to mammals with familial hypercholesterolemia of homozygous form has beneficial effects on plasma cholesterol levels. We can show that the infusion of GH to LDLR deficient animals reduces total plasma and LDL cholesterol levels. This occurs in parallel with a normalization of a suppressed C7xcex1OH enzymatic activity. In addition, we have also found that GH treatment can potentiate the effect of statins and bile acid sequestrants, two important classes of lipid lowering drugs used in established treatment of patients.
By the present invention it is also shown that GH therapy can be used to reduce plasma LDL cholesterol in FH of the homozygous form. Thus GH treatment alone, optionally in combination with established lipid lowering treatment, can be used to treat mammals characterized by a deficiency of LDL receptors. Therefore, GH could become an adjuvant to current therapy of FH-homozygotes.
The present invention is directed to the use of compounds selected from GH, analogues thereof and GH-releasing compounds, optionally in combination with conventional lipid lowering agents, for the preparation of a drug which reduces the serum lipids in a mammal that displays the syndrome of FH of the homozygous form. The therapy may also be combined with established lipid-lowering treatment. The type of GH that can be used includes natural or recombinant GH, and variant molecules of GH or analogues with the common denominator of ultimately being capable to activate a GH receptor signal in cells in the species that displays the syndrome of FH of homozygous form. One example of GH is Genotropin, which is manufactured by Pharmacia and Upjohn. Examples of analogues are compounds that can enhance GH release or interfere with the cellular mechanisms of GH action. Compounds that cause GH release is exemplified by GH-releasing hormone, somatostatin-antagonists, hexarelin, and the Merck growth hormone releasing compound L-692 429. The route of GH administration to mammals can vary and a common way of administration is by injection therapy. Alternative routes of administration may however be applicable in the practice of the present invention. Such alternative routes for GH administration that presently exist or may be developed include oral, nasal, rectal and transdermal GH therapy. The dose of GH to be used for the treatment of the syndrome of FH of homozygous form may vary dependent on the GH used and the species to be treated as well as the clinical evaluation of a patient. In humans, GH would preferably be injected 7 times a week but less frequent injections could be used as long as the criteria of activating a GH receptor signal that results in reduction of cholesterol is met.
One embodiment of the present invention includes the combined treatment with GH and a lipid lowering therapy as LDL apheresis or with compounds preferably selected from the list of lipid lowering drugs that include for example statins, and bile acid sequestrants such as cholestyramine. Today there are five statins available on the market in Sweden, atorvastatin, cerivastatin, fluvastatin, pravastatin and simvastatin.
The dose ranges of these drugs will be the ones recommended by the respective suppliers for clinical use. The daily dose of human GH (such as Genotropin) would preferably be in range from 0.02 to 0.14 IU/kg depending on the responses obtained. The duration of treatment according to the present invention includes GH treatment in a continuous or in a discontinuous fashion with or without the combination of other types of lipid lowering drugs or procedures. The duration of treatment is dependent on the judgement of the responsible physician. It is preferable that the duration of treatment is lasting for several months and that the effect of treatment is monitored at least every half year by analysis of serum cholesterol.
The syndrome of FH suitable for treatment according to the present invention includes homozygous LDLR mutations of any kind that result in deficient LDLR function. Such patients can be identified clinically from an elevated plasma cholesterol value. In a more refined diagnosis the LDLR gene could be sequenced or the pattern of lipoproteins determined.
This invention includes all embodiments disclosed in the appended claims.